Synaptic plasticity, the modulation of synaptic strength and structure with experience, is central to many physiological and pathological states, including learning and memory and a variety of neuropsychiatric diseases. While much research has focused on the contributions of transcription and translation to long-lasting forms of synaptic plasticity, regulated protein degradation through the ubiquitin proteasome pathway (UPP) provides another common means of controlling the protein composition of cells. Because the UPP can regulate protein concentration with exquisite spatial and temporal control, it could readily contribute to aspects of plasticity over variable time domains and in a synapse-specific manner. The aim of this R21 exploratory grant is to test the hypothesis that the UPP functions locally at the synapse to regulate synaptic strength and growth. We have found that inhibition of the UPP with several bath applied proteasome inhibitors in Aplysia sensory-motor cultures produces long-lasting increases in synaptic strength, enhances serotonin-induced plasticity, and leads to the growth of new synaptic contacts between sensory and motor neurons. We now propose to determine whether the UPP at the synapse functions locally to modulate synaptic efficacy and structure. In specific aim 1, we will differentiate between the role of the UPP at the synapse and at the cell body by locally perfusing proteasome inhibitors at the synapse or the soma of cultured Aplysia sensory-motor neurons and measuring the effect on synaptic strength and on serotonin-induced longterm plasticity. The spatial restriction of proteasome inhibition will be monitored using a GFP reporter construct that is normally degraded by the proteasome. In specific aim 2, we will inhibit the proteasome at the synapse and determine the effect on synaptic structure by time-lapse microscopy. The experiments outlined in this proposal will allow us to elucidate a role for the UPP at the synapse during neuronal plasticity. They are consistent with the purpose of the R21 mechanism in that they will also allow us to obtain a sufficient body of data to write an R01 application, whose aims we anticipate will involve identification of the substrates of the UPP that regulate synaptic efficacy and growth, and analysis of whether and how these substrates are modulated by stimuli that produce long-lasting synaptic plasticity.